Screening method for identifying women at increased risk for imminent delivery

ABSTRACT

The present invention provides an early, biochemical indication of increased risk of impending delivery. The method is particularly useful to identify those pregnant women who are at increased risk for preterm delivery and can also be used to identify those women at risk for a post-date delivery. The method comprises obtaining a cervicovaginal secretion sample from a pregnant patient after week 12 of gestation and determining the level of total fibronectin in the sample. The presence of an elevated fibronectin level in the sample indicates an increased risk of delivery. The test detects greater than 80% of women who deliver prematurely, as early as two to three weeks prior to delivery.

This application is a continuation of application Ser. No. 07/787,271,filed Nov. 4, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for detection of impending delivery,particularly impending preterm delivery. In particular, this inventionis directed to the determination of impending delivery by detecting anincreased level of total fibronectin in cervicovaginal secretionsamples.

2. Description of the Prior Art

Determination of impending preterm births is critical for increasingneonatal survival of preterm infants. In particular, preterm neonatesaccount for more than half, and maybe as much as three-quarters of themorbidity and mortality of newborns without congenital anomalies.Although tocolytic agents which can delay delivery were introduced 20 to30 years ago, there has been only a minor decrease in the incidence ofpreterm delivery. It has been postulated that the failure to observe alarger reduction in the incidence of preterm births is due to errors inthe diagnosis of preterm labor and to the patients' conditions being tooadvanced for tocolytic agents to successfully delay the birth.

Traditional methods of diagnosis of preterm labor have highfalse-negative and false-positive error rates [Friedman et al, Am. J.Obstet. Gynecol. 104:544 (1969)]. In addition, traditional methods fordetermining impending preterm delivery, particularly in patients withclinically intact membranes, may require subjective interpretation, mayrequire sophisticated training or equipment [Garl et al, Obstet.Gynecol. 60:297 (1982)] or may be invasive [Atlay et al, Am. J. Obstet.Gynecol. 108:933 (1970)]. An early, objective biochemical marker whichindicated increased risk for preterm delivery was sought.

Although preterm neonates account for the majority of the morbidity andmortality of newborns without congenital anomalies, post-date deliveries(deliveries after the due date of the baby) are also a significantsource of morbidity and mortality. An early method of detectingimpending delivery could also identify women late in gestation who areabout to deliver and thus will not deliver post-date.

Recently, Lockwood et al [New Engl. J. Med., 325:669-674 (1991)]reported that fetal fibronectin in cervical and vaginal secretionsindicates pregnancies which are at risk of imminent delivery. Theauthors postulate that damage to the fetal membranes may release fetalfibronectin into the cervix and vagina, giving rise to the biochemicalmarker.

Other markers which may be released in women with true threatenedpregnancies can be used to screen those women who should be closelymonitored and to provide additional information about the stage of thedisease.

SUMMARY OF THE INVENTION

The present invention provides an early, biochemical indication ofincreased risk of impending delivery. The method is particularly usefulto identify those pregnant women who are at increased risk for pretermdelivery. However, the method can also be used to detect those women whoare at risk for post-date delivery. The method comprises obtaining acervicovaginal secretion sample from a pregnant patient after about week12 of gestation and determining the level of total fibronectin in thesample. The presence of an elevated fibronectin level in the sampleindicates an increased risk of imminent delivery. The test is both asensitive and specific screen for pregnancies at risk and can detectimpending delivery about two to three weeks prior to delivery.

The test is preferably administered to women at about 12 weeks gestationand repeated at each perinatal visit (every two to four weeks) until atleast week 37, preferably until delivery, if the test is negative. Forthose patients whose assay result indicates an increased risk of pretermdelivery, a test of the patient's fetal fibronectin level can be made toconfirm the increased risk and to estimate how soon the delivery may be.In addition, those patients can be carefully monitored, as are otherpatients at risk.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a screening assay which provides an early,biochemical indication of increased risk of imminent delivery. Themethod can provide an indication of impending delivery as early as twoto three weeks prior to delivery. This method allows early interventionin the course of preterm delivery and provides an additional factorwhich can indicate those pregnancies at greatest risk. The method canalso indicate those women late in gestation who are not likely todeliver in the next few weeks. Appropriate action to monitor orintervene in the impending post-date delivery can be taken.

The method comprises obtaining a cervicovaginal secretion sample fromthe vaginal cavity or the external cervical canal from a pregnantpatient after about week 12 of pregnancy and determining the level offibronectin in the sample. The presence of an elevated fibronectin levelin the sample indicates a patient who is at risk for imminent delivery.

The present method can determine impending delivery from early ingestation through post-date deliveries. Deliveries prior to 20 weeksgestation are called spontaneous abortions rather than pretermdeliveries. The present method can be used to detect spontaneousabortions (12 to 20 weeks gestation), preterm deliveries (20 to 37 weeksgestation), term deliveries (37 to 40 weeks gestation) and post-datedeliveries (deliveries after 40 weeks gestation).

Patients to be Tested

The present method can be used on any pregnant woman following about 12weeks gestation. In addition to screening any woman to determine whetherdelivery is imminent, the patients who should be screened are thosepatients with clinically intact membranes in a high risk category forpreterm delivery, and preferably, all those women whose pregnancies arenot sufficiently advanced to ensure delivery of a healthy fetus. Ninetypercent of the fetal morbidity and 100 percent of the fetal mortalityassociated with preterm delivery is for those fetuses delivered prior to32 to 34 weeks gestation. Therefore, 32 to 34 weeks gestation is animportant cutoff for the health of the fetus, and women whosepregnancies are at least about 12 weeks and prior to 34 weeks ingestation should be tested.

In addition there are a large number of factors known to be associatedwith the risk of preterm delivery. Those factors include multiple fetusgestations; incomplete cervix; uterine anomalies; polyhydramnios;nulliparity; previous preterm rupture of membranes or preterm labor;preeclampsia; first trimester vaginal bleeding; little or no antenatalcare; and symptoms such as abdominal pain, low backache, passage ofcervical mucus and contractions. Any pregnant woman at 12 or more weeksgestation with clinically intact membranes and having one or more riskfactors for preterm delivery should be tested throughout the riskperiod; i.e., until about week 37. Risk factors for spontaneous abortioninclude gross fetal anomalies, abnormal placental formation, uterineanomalies and maternal infectious disease, endocrine disordercardiovascular renal hypertension, autoimmune and other immunologicdisease, and malnutrition.

In addition, any woman who is late in gestation and may deliverpost-date should be tested. Those patients include women after aboutweek 37 through delivery. Risk factors for post-date deliveries includemultiparity, maternal obesity, fetal macrosomia, advanced maternal age,previous post-date delivery, male fetus, and certain genetic disorders.

Sample

The sample is obtained in the vicinity of posterior fornix, theectocervix or external cervical os. The sample generally comprises fluidand particulate solids, and may contain vaginal or cervical mucus andother vaginal or cervical secretions. The sample is preferably removedwith a swab having a dacron or other fibrous tip. Alternatively, thesample can be obtained with a suction or lavage device. Calculations toaccount for any additional dilution of the samples collected usingliquids can be performed as part of the interpretation of the assayprocedure.

Following collection, the sample is transferred to a suitable containerfor storage and transport to a testing laboratory. It is important thatthe sample be dispersed in a liquid which preserves proteinaceousanalytes. The storage and transfer medium should minimize, preferablyprevent, decline in the analyte level during storage and transport. Asuitable solution for storage and transfer consists of 0.05M Tris-HCl,pH 7.4; 0.15M NaCl, 0.02% NAN₃, 1% BSA, 500 Kallikrein Units/ml ofaprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM EDTA, andis described in U.S. Pat. No. 4,919,889, issued Apr. 24, 1990. Thesolution is also suitable as a sample diluent solution.

Alternatively, home and office use devices for immediate processing ofthe sample can be used. If used, the sample is placed directly in thedevice and testing is performed within minutes of sample collection. Insuch cases, the need to stabilize the analyte is minimized and anysolution which facilitates performing the assay and is not detrimentalto analyte stability can be used.

Assay Procedure

Fibronectin is assayed by any procedure which can determine the presenceof a threshold quantity of fibronectin in the sample. Immunoassays arepreferred. The antibody affinity required for detection of fibronectinusing a particular immunoassay method will not differ from that requiredto detect other polypeptide analytes. The antibody composition can bepolyclonal or monoclonal.

Anti-fibronectin antibodies can be produced by a number of methods.Polyclonal antibodies can be induced by administering an immunogeniccomposition comprising human fibronectin to a host animal. Thefibronectin is preferably plasma fibronectin rather than cellularfibronectin or fetal fibronectin. Both the cellular and fetal isoformscontain extra domains (additional unique amino acid regions which arenot present in plasma fibronectin). Thus, antibodies to those uniqueregions will not react with all fibronectin isoforms which may bepresent in the sample.

Preparation of immunogenic compositions of fibronectin may varydepending on the host animal and is well known. For example, fibronectinor an antigenic portion thereof can be conjugated to an immunogenicsubstance such as KLH or BSA or provided in an adjuvant or the like. Theinduced antibodies can be tested to determine whether the composition isfibronectin-specific. If a polyclonal antibody composition does notprovide the desired specificity, the antibodies can be purified toenhance specificity by a variety of conventional methods. For example,the composition can be purified to reduce binding to other substances bycontacting the composition with fibronectin affixed to a solidsubstrate. Those antibodies which bind to the substrate are retained.Purification techniques using antigens affixed to a variety of solidsubstrates such as affinity chromatography materials including Sephadex,Sepharose and the like are well known.

Monoclonal fibronectin-specific antibodies can also be prepared byconventional methods. A mouse can be injected with an immunogeniccomposition comprising fibronectin, and spleen cells obtained. Thosespleen cells can be fused with a fusion partner to prepare hybridomas.Antibodies secreted by the hybridomas can be screened to select ahybridoma wherein the antibodies react with fibronectin and exhibitsubstantially no reaction with the other proteins which may be presentin a sample. Hybridomas that produce antibodies of the desiredspecificity are cultured by standard techniques. Hybridoma preparationtechniques and culture methods are well known and constitute no part ofthe present invention.

An exemplary preparation of polyclonal antibodies is described in theexamples. Antibody preparation and purification methods are described ina number of publications including Tijssen, P. Laboratory Techniques inBiochemistry and Molecular Biology: Practice and Theories of EnzymeImmunoassays New York: Elsevier (1985), for example.

A number of different types of immunoassays are well known using avariety of protocols and labels. The assay conditions and reagents maybe any of a variety found in the prior art. The assay may beheterogeneous or homogeneous, conveniently a sandwich assay.

The assay usually employs solid phase-affixed anti-fibronectinantibodies. The antibodies may be polyclonal or monoclonal. The solidphase-affixed antibodies are combined with the sample. Binding betweenthe antibodies and sample can be determined in a number of ways. Complexformation can be determined by use of soluble antibodies specific forfibronectin. The antibodies can be labeled directly or can be detectedusing labeled second antibodies specific for the species of the solubleantibodies. Various labels include radionuclides, enzymes, fluorescers,colloidal metals or the like. Conveniently, the assay will be aquantitative enzyme-linked immunosorbent assay (ELISA) in whichpolyclonal antibodies specific for fibronectin are used as the solidphase-affixed and enzyme-labeled, soluble antibodies. Alternatively, theassay can be based on competitive inhibition, where fibronectin in thesample competes with a known amount of fibronectin for a predeterminedamount of anti-fibronectin antibody. For example, any fibronectinpresent in the sample can compete with a known amount of the labeledfibronectin or fibronectin analogue for antibody binding sites. Theamount of labeled fibronectin affixed to the solid phase or remaining insolution can be determined.

In another preferred embodiment, the assay is a homogeneous immunoassayin which polyclonal antibodies specific for fibronectin are used as thesolid phase-affixed and colloidal metal-labeled, soluble antibodies.Appropriate dilution of the conjugate can be performed to detect theselected threshold level of fibronectin as a positive sample.

Threshold Value

The amount of total fibronectin in the sample is significantly elevatedover the level for pregnant women at the same stage of gestation withnormal pregnancies. Preferably, the total fibronectin concentration inthe sample is at least about 600 to 750 ng/ml of sample after week 20 ofgestation. Between weeks 12 and 20 the threshold values vary. Selectedthreshold values for the earliest portion of gestation are described indetail in the examples.

It is difficult to quantify proteins in vaginal swab samples for tworeasons. The first is that the amount of fluid collected by the swabvaries. The second is the total volume of secretions present in thecervicovaginal region varies. Therefore, any measurement of thefibronectin concentration in a particular sample is only asemi-quantitative indication of the total amount of fibronectin in thecervicovaginal region. Therefore, it is desirable that patients withsamples near the threshold value be retested in a follow up visit.

A study determining that a threshold value after week 20 of gestation inthe range of 600 to 750 ng/ml of sample is an indication of risk isdescribed in detail in the examples.

Diagnostic screening methods to detect disease in the general populationare conventionally designed to give very high sensitivity (detecting allthose with disease) while sacrificing specificity (falsely detectingsome who are free of disease). Because specificity is expected to be lowin screening protocols, alternative (and usually more costly andsophisticated) diagnostic methods are typically used to confirm theexistence of disease. Therefore, variations on the above thresholdvalues can be made to make the assay either more specific or moresensitive.

Interpretation of Assay Result

Elevated levels of total fibronectin indicate increased risk of pretermdelivery. As explained in detail in the examples, the assay is sensitiveand specific. In addition, the assay has a high negative predictivevalue. That means that a large percentage of patients who deliveredearly had an elevated fibronectin value. Since the test successfullydetects a large percentage of patients who deliver early, the test is aneffective screening procedure for women at risk of a preterm deliverywho do not have any other risk indicators.

The test can be administered to any pregnant woman following about 12weeks gestation until delivery, or at least until the risk of prematuredelivery (i.e., until about week 37) ceases. Preferably, it isadministered to all women with any known risk factor following 12 weeksgestation until delivery.

If the total fibronectin test is positive (above the threshold value),the patient is preferably tested for the presence of fetal fibronectinin her cervicovaginal secretions. If fetal fibronectin is present in thesecretions, the patient is likely to deliver in two to three days.Measures to determine or enhance fetal lung maturity can be undertaken.If the fetal fibronectin assay is negative, the patient should becarefully monitored and repeated evaluations of the patient's fetalfibronectin levels should be performed on subsequent visits. In general,patients at risk for preterm delivery are examined every two weeks fromabout 22 to 36 weeks, rather than every four weeks as for patients in alow risk category. All patients are examined weekly from about week 36.

If the total fibronectin test is negative, the test is preferablyrepeated on each subsequent antenatal visit until either the test ispositive or the patient has delivered her baby.

This invention is further illustrated by the following specific butnon-limiting examples. Temperatures are given in degrees Centigrade andconcentrations as weight percent unless otherwise specified. Procedureswhich are constructively reduced to practice are described in thepresent tense, and procedures which have been carried out in thelaboratory are set forth in the past tense.

EXAMPLE 1 Quantitation of Total Fibronectin in a Vaginal Swab Sample

An immunoassay to determine total fibronectin in a vaginal sample usedthe reagents and procedures described below.

Preparation of Polyclonal Anti-Human Fibronectin Antibody

Human plasma fibronectin was purified from human plasma as described byEngvall and Ruoslahti, Int. J. Cancer 20:1-5 (1977). The anti-humanplasma fibronectin antibodies were elicited in goats using theimmunization techniques and schedules described in the literature, e.g.,Stollar, Meth. Enzym. 70:70 (1980), immunizing the goats with the humanplasma fibronectin antigen. The antiserum was screened in a solid phaseassay similar to that used for monoclonal antibodies, e.g., as describedby Lange et al, Clin. Exp. Immunol. 25:191 (1976) and Pisetsky et al, J.Immun. Meth. 41:187 (1981).

The IgG fraction of the antiserum was purified further by affinitychromatography using CNBr-Sepharose 4B (Pharmacia Fine Chemicals) towhich has been coupled human plasma fibronectin according to the methodrecommended by the manufacturer (AFFINITY CHROMATOGRAPHY, Pharmacia FineChemicals Catalogue 1990), pp 15-18.

Briefly, the column was equilibrated with from 2 to 3 volumes of buffer(0.01 M PBS, pH 7.2), and the anti-human fibronectin antibody-containingsolution was then applied to the column. The absorbency of the effluentwas monitored at 280 nm until protein no longer passed from the column.The column was then washed with equilibration buffer until a baselineabsorbance at 280 nm was obtained.

The immunoaffinity bound anti-human plasma fibronectin antibody waseluted with 0.1M glycine buffer, pH 2.5. Peak protein fractions werecollected, pooled and dialyzed against 0.01M PBS, pH 7.2, for 24-36 hrat 4° C. with multiple buffer changes.

The above procedure was repeated to immunize rabbits with human plasmafibronectin and to purify the resultant polyclonal anti-humanfibronectin antibodies.

Preparation of Anti-Fibronectin Antibody-Coated Microtiter Plate

Goat anti-human plasma fibronectin prepared as described above wasdiluted to 10 μg/ml in 0.05M carbonate buffer, pH 9.6. 100 μl wasdispersed into each well of a polystyrene microtiter plate such assupplied by Costar, Nunc, or Dynatech. The plate was covered andincubated 2 to 4 hr at room temperature or 4° C. overnight. The platewas washed 3 to 4 times with Wash Buffer (0.02M Tris HCl, 0.015M NaCl,0.05% TWEEN-20), filling and emptying the wells completely with eachuse. The plate was then blocked by dispersing 200 μl of ablocking/stabilizing solution (4% sucrose, 1% mannitol, 0.01M PBS, 1%BSA, 0.02% NAN₃, pH 7.4) into each well and incubating for 30 minutes to2 hrs at room temperature. The wells were then aspirated to dryness, theplate was packaged in an air-tight container with a desiccant pouch, andstored at 4° C. until needed. The wells were present as eight wellstrips.

Preparation of Enzyme Labeled Anti-(fibronectin) Antibody

Anti-human plasma fibronectin antibody prepared as described above wasconjugated with alkaline phosphatase following the one-stepglutaraldehyde procedure of Avrameas, Immunochem. 6:43 (1969).

Assay Reagents

The assay was performed using the following additional reagents. Thestock antibody conjugate was appropriately diluted in conjugate diluent(0.05M Tris Buffer pH 7.2, 2% D-Sorbitol, 2% BSA, 0.1% Sodium Azide,0.01% Tween-20, 1 mM Magnesium Chloride, and 0.1% Zinc Chloride) and 10ml placed in a polyethylene dropper bottle container.

The enzyme substrate (10 ml in a polyethylene dropper bottle container)was phenolphthalein monophosphate (1 mg/ml) dissolved in 0.4Maminomethylpropanediol buffer, pH 10 with 0.1 mM magnesium chloride and0.2% sodium azide.

The fibronectin calibration solutions were plasma fibronectin(fibronectin from human serum purchased from Boehringer Mannheim,Indianapolis, Ind.; Catalogue No. 1050407) diluted to a concentration of0.0, 0.01, 0.05 and 0.25 μg/ml in sample diluent solution (0.05M Trisbuffer pH 7.4, 1% bovine serum albumin (BSA), 0.15M sodium chloride,0.02% Sodium Azide, 5 mM ethylenediamine tetraacetic acid (EDTA), 1 mMphenylmethylsulfonyl fluoride (PMSF), and 500 Kallikrein Units/ml ofAprotinin). This sample diluent solution is described in U.S. Pat. No.4,919,889 to Jones et al, issued Apr. 24, 1990, which patent isincorporated herein by reference in its entirety. The negative controlwas the sample diluent solution used for the positive control withoutfibronectin.

The rinse buffer (10 ml in a polyethylene dropper bottle container) wasa 50X concentrate containing 1.0M Tris buffer pH 7.4, 4.0M sodiumchloride, 2.5% Tween-20, and 1% sodium azide. The rinse buffer wasdiluted with water to a final concentration of 0.02M Tris, 0.08M sodiumchloride, 0.05% Tween-20, and 0.02% sodium azide for use in the assay.

In addition, 5μ pore size polyethylene sample filters (PorexTechnologies, Fairburn, Ga.) were used to filter the samples prior toassay. All of the dropper bottles used to perform the assay werepolyethylene bottles designed to dispense approximately 50 μl drops ofthe reagent. All of the assay steps performed following samplecollection utilized the reagents and materials described above.

Assay Procedure

The assay was performed as follows. All samples were collected in thevicinity of the posterior fornix or cervical os using dacron swabs. Swabsamples were immersed in 1.0 ml of sample diluent in a collection vial.The swabs were removed from the solution leaving as such liquid aspossible in the collection tube. The samples were incubated at 37° C.along with the controls for 15 minutes prior to the assay, either beforeor after filtration. A sample filter was snapped in place on each sampletube. The 8-well strips were snapped into place in a strip holder. Theholder had the alphanumeric indications of the 12 columns and eight rowsof standard microtiter plates. Duplicate 100 μl aliquots of each sampleand the positive and negative controls were placed in separate wells ofthe microtiter strip and incubated for 1 hour at room temperature.

Following incubation, samples and controls were aspirated from thewells. Wells were washed three times with diluted wash buffer (1X).Following washing, 100 μl of enzyme-antibody conjugate was added to eachwell and incubated for 30 minutes at room temperature. The wells wereaspirated and washed as described above. Following washing, 100 μl ofenzyme substrate was added to each well and incubated for 30 minutes atroom temperature.

Following the incubation, the plates were gently agitated by hand orwith an orbital shaker to mix the well contents. The frame of strips wasplaced in an ELISA plate reader. The absorbance of each well at 550 nmwas determined. The average absorbance of the duplicate wells for eachsample and control was calculated. The total fibronectin concentrationfor the samples was calculated by preparing a standard curve andestimating that the samples were diluted to about one-tenth of theiroriginal concentration (collection of about 0.1 ml of sample combinedwith 1.0 ml of diluent).

EXAMPLE 2 Detection of a Threshold Amount of Total Fibronectin in aVaginal Swab Sample

In another preferred embodiment, an assay kit to detect a thresholdamount of fibronectin includes the following components. This kit isdesigned to be used to perform a rapid, bedside or doctor's officeassay.

1. an assay device comprising a plastic housing and containing:

(a) a porous nylon membrane to which is bound an anti-fibronectinantibody;

(b) a flow control membrane system; and

(c) an absorbent layer

2. a colloidal gold-labeled goat anti-fibronectin antibody conjugate ina protein matrix

3. conjugate reconstitution buffer

4. a wash solution

5. a sterile, dacron sample collection swab

The membrane device is prepared by the following procedure.Approximately 2 μl of the polyclonal anti-fibronectin antibody preparedas described in Example 1 is applied to a membrane surface (1.2μ nylon,Biodyne-A, Pall) in a pH 6, 0.01M phosphate buffered saline (PBS), 0.1 Mcitrate buffer containing 0.5 mg/ml BSA. A procedural control consistingof human plasma fibronectin purified as described in Example 1 in thesame buffer is also applied to a discrete region of the membrane. Afterthe membrane has air dried, a blocking reagent of PBS-buffered, 0.5%nonfat dry milk is added to the membrane. The excess blocking reagent isremoved after at least about 20 minutes.

The membrane-holding device (Target Device, V-Tech, Pomona, Calif.) isassembled with a second porous layer (0.45μ low protein-binding nylon,LoProdyne, Pall) beneath the antibody-bearing membrane (in the directionof sample application) for controlling the flow of sample solution fromthe assay membrane to the absorbent layer. The two porous membranes arethen placed over an absorbent porous polyethylene layer having acapacity of greater than 1.5 ml (Chromex, Brooklyn, N.Y.) and enclosedin the device. The device is packaged individually in a sealed plasticbag containing desiccant.

The colloidal gold was prepared by the reduction of 0.01%tetrachloroauric acid with 0.16% sodium citrate in a manner whichproduces approximately 30 nm particles. Briefly, the two solutions areheated separately to 90° C. The reducing solution is added to the goldsolution while vigorously stirring. The combined solution is boiled(100° C.) for at least 10 minutes.

Affinity purified goat anti-fibronectin antibody (prepared as describedin Example 1) was bound to the colloidal gold by adsorption. Briefly,the colloidal gold solution prepared above was combined with theantibody (5-10 μg/ml) in water. Following conjugation, the conjugatesolution is stabilized by the addition of 5% BSA and 5%polyvinylpyrrolidone (final concentration).

The stock conjugate was concentrated approximately 10- to 12-fold byultrafiltration using a hollow fiber filter. The concentrated conjugatewas diluted to an appropriate level in 15 mM Tris, 2% BSA, 0.1% Tween20, 0.2% polyethylene glycol, 8% polyvinylpyrrolidone and 0.04%thimerosal. An appropriate concentration is determined by using a rangeof dilutions in a sample assay procedure as described below anddetermining the dilution which produces the best result. This titrationprocedure is used to set the threshold detection level for totalfibronectin at 750 ng/ml.

The selected conjugate dilution is placed in polyethylene samplecollection tubes and lyophilized. The tubes are fitted with 2μ pore sizepolyethylene sample filters (Porex Technologies, Fairburn, Ga.) duringthe lyophilization process. The lyophilized conjugate is individuallypackaged in a foil pouch with desiccant.

The conjugate reconstitution buffer is 100 mM sodium acetate. Thisbuffer is packaged as a unit dose in a 1 ml disposable tube. The washsolution is water packaged as a unit dose in a disposable tube. The kitadditionally contains an individually packaged sterile dacron swab and aprocedural summary card.

The assay was performed as follows.

1. Before collecting the sample, remove the plastic tube containing goldconjugate from the foil pouch, remove the dropper tip and add the entirecontents of the tube containing the conjugate reconstitution buffer.

2. Collect the sample with the swab provided. During a sterile speculumexamination, insert the swab into the posterior fornix of the vagina,twirl for approximately 10 seconds to absorb fluid. Immediately proceedto perform the test. Samples may not be stored for later testing. Placethe swab in the gold conjugate solution and mix rapidly with an up anddown motion for 10 to 15 seconds.

3. Remove as much liquid as possible from the swab by rolling the tip onthe inside of the tube. Dispose of the swab in a manner consistent withhandling potentially infectious materials.

4. Replace the dropper tip on the plastic tube and immediately dispensethe entire volume of diluted filtered sample onto the surface of themembrane device.

5. After the sample liquid has been absorbed into the membrane surface,add a few drops of wash solution and observe the results.

6. A negative result is indicated by a red color in the proceduralcontrol area of the membrane only. A positive result is indicated by apink or red spot in the test zone of the membrane as well as in thecontrol zone.

EXAMPLE 3 Total Fibronectin Levels in Preterm Patients

In an effort to evaluate cervicovaginal expression of total fibronectinas a screen for preterm delivery (preterm delivery), 73 asymptomaticwomen with elevated risk for preterm delivery were identified andfollowed longitudinally. Factors which defined these women as havinghigher risk included pregnancy with twins, uterine anomalies, previouspreterm labor and previous preterm delivery. Vaginal specimens wereobtained from these women at 1 to 2 week intervals between 24 and 34weeks gestation. Vaginal secretions were collected from the ectocervicalregion of the external cervical os and the posterior fornix of thevagina using separate dacron swabs.

The total fibronectin concentration of vaginal secretion samples in thisstudy was determined as described in Example 1. The averageconcentration of total fibronectin in cervicovaginal secretions was4.49±0.50 μg/ml (mean±SEM) for women with uncomplicated pregnanciesprior to 22 weeks gestation. The concentration of total fibronectinexceeded 750 ng/ml in 64.8% (250/386) of these women. After 22 weeksgestation the average concentration of total fibronectin was 0.99±0.50and 22.2% (4/18) had values greater than 750 ng/ml.

From those values, a sample was determined to be positive if its totalfibronectin concentration exceeded 750 ng/ml. A single positive samplewas the minimal requirement for definition of a positive patient. Thesample results for all women were accumulated and compared togestational age at the time of delivery. Delivery prior to 37 weeks and0 days was defined as preterm while a pregnancy exceeding 37 days and 0days was defined as term.

Of the 73 patients enrolled in this evaluation, 24 delivered prematurelyand 49 delivered at term. The relationship of total fibronectin resultsand clinical outcome is shown in Table 1. In the analyses, sensitivitymeans the number of true positive test results divided by the totalnumber of women with the condition; i.e., the number of true positivetest results divided by the sum of the number of true positive and falsenegative test results). Specificity means the number of true negativetest results divided by the total number of women without the condition;i.e., the number of true negative test results divided by the sum of thenumber of true negative and false positive test results. Positivepredictive value (PV+) means the number of true positive test resultsdivided by the total number of samples which tested positive. Negativepredictive value (PV-) means the number of true negative test resultsdivided by the total number of samples which tested negative.Sensitivity, specificity, positive predictive value and negativepredictive value are based on detection of preterm delivery (PTD),rather than term delivery (TD) in this analysis.

    ______________________________________                                               FN +         FN -                                                      ______________________________________                                        PTD      19              5      24                                            TD       24             25      49                                                     43             30      73                                            ______________________________________                                         Sensitivity = 82.8%?                                                          Specificity = 51.0%?                                                          PV + = 44.2%?                                                                 PV - = 83.3%?                                                                 Relative Risk = 2.65, p < 0.007?                                              MantelHaenszel X.sub.2 = 5.98, p < 0.014                                 

At least one total fibronectin sample exceeded 750 ng/ml in 19 of 24patients (sensitivity=82.8%) delivering prematurely and 24 of 49patients delivering at term (specificity=51.0%). Alternatively, thepredictive value of a positive test was 44.2% while the predictive valueof a negative test was 83.3%. The relative risk associated with apositive test (relative to a negative test) was 2.65 which issignificantly different from 1.00 (Null Hypothesis (H_(o) : RelativeRisk=1.00) with a p value of less than 0.007. Moreover, the distributionof positive results was associated with preterm delivery as shown by theMantel-Haenszel X₂ test statistic of 5.98 (p<0.014).

The results of the study demonstrate that the test was sensitive andacceptably specific for screening in that most women who deliver earlyare detected by the test.

EXAMPLE 4 Total Fibronectin Levels in Weeks 12 to 22

Total fibronectin concentrations were determined in specimens ofcervicovaginal secretions obtained from women with apparentlyuncomplicated pregnancies between 12 weeks gestation and term asdescribed in Example 1. Average concentrations of total fibronectin weredetermined at each gestational age.

Total fibronectin concentration was highest at 12 weeks gestation andgradually decreased until week 22. From week 22 of gestation until nearterm, total fibronectin concentration rarely exceeded 500 ng/ml. Asgestational age advanced beyond 34 weeks, total fibronectinconcentrations gradually increased presaging delivery. The gestationalage dependency of total fibronectin concentration was accuratelypredicted by a series of second order polynomial regression equationscalculated using estimated gestational age at sampling and totalfibronectin concentration as the independent and dependent variables,respectively.

The predicted concentrations of total fibronectin (μg/ml) at each weekof gestation are shown in the following table.

    ______________________________________                                        Gestational                                                                             Prod         Gestational                                                                             Pred                                         Age       [Total FN]   Age       [Total FN]                                   ______________________________________                                        12        6.30         25        0.10                                         13        6.20         26        0.06                                         14        5.94         27        0.03                                         15        5.51         28        0.04                                         16        4.92         29        0.07                                         17        4.16         30        0.13                                         18        3.24         31        0.22                                         19        2.16         32        0.33                                         20        0.91         33        0.47                                         21        0.57         34        0.64                                         22        0.42         35        0.83                                         23        0.29         36        1.06                                         24        0.18         37        1.31                                                                38        1.58                                         ______________________________________                                    

What is claimed is:
 1. An immunoassay method of screening for anincreased risk of impending delivery comprising:a) obtaining a secretionsample from the vaginal cavity or the cervical canal from a pregnantpatient after week 12 of pregnancy; and b) determining the level oftotal fibronectin in the sample, wherein an elevated level of totalfibronectin in the sample suggests an increased risk of impendingdelivery.
 2. The method of claim 1 wherein the sample is obtained fromthe posterior fornix.
 3. The method of claim 1 wherein the sample isobtained from the cervical os.
 4. The method of claim 1 wherein thesample obtained from the patient is determined not to have an elevatedtotal fibronectin level and the method further comprises repeating thesteps of the method with at least one other sample from the patientobtained at least two weeks after the sample determined not to have anelevated total fibronectin level was obtained.
 5. The method of claim 4wherein said other sample is obtained 2 weeks to about 4 weeks afterobtaining the sample and the steps of the method are repeated with asuccession of one or more other samples until the patient delivers or asample determined to have an elevated total fibronectin level isobtained.
 6. The method of claim 1 wherein the sample is determined tohave an elevated total fibronectin level and the method furthercomprises performing a second immunoassay to determine the presence offetal fibronectin in a sample of cervicovaginal secretions obtained fromthe patient wherein determination of the presence of fetal fibronectinby said second immunoassay confirms the increased risk of impendingdelivery.
 7. The method of claim 6 wherein the sample of cervicovaginalsecretions is determined not to contain fetal fibronectin and the methodfurther comprises repeating the determination of fetal fibronectin withat least one other sample of cervicovaginal secretions from the patientobtained at least one week after the sample determined not to containfetal fibronectin was obtained.
 8. The method of claim 7 wherein saidother sample of cervicovaginal secretions is obtained 2 weeks afterobtaining the sample and the determination of fetal fibronectin isrepeated with a succession of one or more other samples obtained at twoweek intervals until the patient delivers or a sample positive for fetalfibronectin is obtained.
 9. The method of claim 1 wherein thedetermining step comprises the steps of:a) contacting the sample with afirst anti-(total fibronectin) antibody for a time sufficient forantigen-antibody binding; and b) determining the amount of binding todetermine the level of total fibronectin in the sample.
 10. The methodof claim 9 wherein said first anti-(total fibronectin) antibody isaffixed to a solid phase and the amount of binding is determined by:a)contacting the sample with a second anti-(total fibronectin) antibodyfor a time sufficient for antigen-antibody binding, said secondanti-(total fibronectin) antibody being directly or indirectly labeled;and b) determining the amount of label on said second antibodyspecifically bound to the solid phase to determine the level of totalfibronectin in the sample.
 11. The method of claim 10 wherein thelabeled anti-(total fibronectin) antibody is conjugated to a label. 12.The method of claim 11 wherein the label is an enzyme.
 13. The method ofclaim 10 wherein the labeled anti-(total fibronectin) antibody isindirectly labeled by reacting the anti-(total fibronectin) antibodywith a labeled antibody specific for the anti-(total fibronectin)antibody.